Method of Using FOXO3A Polymorphisms and Haplotypes to Predict and Promote Healthy Aging and Longevity

ABSTRACT

The invention provides methods and compositions relating to identification and use of genetic information from the FOXO3A gene that can be used for determining and increasing an individual&#39;s likelihood of longevity and of retaining physical and cognitive function during aging, and for determining and decreasing an individual&#39;s likelihood of developing a cardiovascular-, metabolic- or age-related disease, including coronary artery (heart) disease, stroke, cancer, chronic pulmonary disease, diabetes, Parkinson&#39;s disease and dementia.

CROSS-REFERENCE TO RELATED APPLICATION

This application is in part based on, and claims the benefit of U.S.Provisional Patent Application No. 61/087,722, filed Aug. 10, 2008,which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant 1 R01AG027060-01 (Defining the Healthy Aging Phenotype) from the NationalInstitute on Aging. Additional funding was provided by U.S. governmentsupport under contract N01-HC-05102 from the National Heart, Lung, andBlood Institute, contract N01-AG-4-2149 and grants 5 U01 AG019349-05 andK08 AG22788-02 from the National Institute on Aging. The U.S. governmenthas certain rights in the invention. Additional support came under grant2004-0463 from the Hawaii Community Foundation.

FIELD OF INVENTION

The invention relates to a method of using FOXO3A polymorphisms andhaplotypes in diagnostics to predict or in planning treatments andinterventions to promote healthy aging and longevity.

BACKGROUND OF THE INVENTION

The FOXO3A gene belongs to the forkhead family of transcription factorswhich are characterized by a distinct forkhead domain. This gene likelyfunctions as a trigger for apoptosis through expression of genesnecessary for cell death. Translocation of this gene with the MLL geneis associated with secondary acute leukemia. Alternatively splicedtranscript variants encoding the same protein have been observed.

The FOXO3A gene is one of the human homologs of DAF-16, a gene that hasbeen described to extend lifespan in the model organisms C. elegans(Murphy C T (2006) The search for DAF-16/FOXO transcriptional targets:approaches and discoveries. Exp Gcrontol 41:910-921) and D.melanogaster. (Giannakou M E et al. (2007) Dynamics of the action ofdFOXO on adult mortality in Drosophila. Aging Cell 6:429-438).

The FOXO3A gene is located on human chromosome 6q21, from position108,987,719 to 109112664 (NCBI ver. 36), is composed of four (4) exonsthat can be alternately expressed, which results in the same protein(variant #1 is described by file NM_(—)001455.3; variant #2 is describedby file NM_(—)201559.2. The FOXO3A protein is composed of 673 aminoacids and is 71,277 Da in size. The amino acid sequence of FOXO3A, asdefined by the file “NP_(—)963853” at the NCBI is identified as SEQ IDNo. 1 and is the following:

MAEAPASPAPLSPLEVELDPEFEPQSRPRSCTWPLQRPELQASPAKPSGETAADSMIPEEEDDEDDEDGGGRAGSAMAIGGGGGSGTLGSGLLLEDSARVLAPGGQDPGSGPATAAGGLSGGTQALLQPQQPLPPPQPGAAGGSGQPRKCSSRRNAWGNLSYADLITRAIESSPDKRLTLSQTYEWMVRCVPYFKDKGDSNSSAGWKNSIRHNLSLHSRFMRVQNEGTGKSSWWIINPDGGKSGKAPRRRAVSMDNSNKYTKSRGRAAKKKAALQTAPESADDSPSQLSKWPGSPTSRSSDELDAWTDFRSRTNSNASTVSGRLSPIMASTELDEVQDDDAPLSPMLYSSSASLSPSVSKPCTVELPRLTDMAGTMNLNDGLTENLMDDLLDNITLPPSQPSPTGGLMQRSSSFPYTTKGSGLGSPTSSFNSTVFGPSSLNSLRQSPMQTIQENKPATFSSMSHYGNQTLQDLLTSDSLSHSDVMMTQSDPLMSQASTAVSAQNSRRNVMLRNDPMMSFAAQPNQGSLVNQNLLHHQHQTQGALGGSRALSNSVSNMGLSESSSLGSAKHQQQSPVSQSMQTLSDSLSGSSLYSTSANLPVMGHEKFPSDLDLDMFNGSLECDMESIIRSELMDADGLDFNFDSLISTQNVVGLNVGNFTGAKQASSQSWVPG

FOXO3A interacts with YWHAB/14-3-3-beta and YWHAZ/14-3-3-zeta, UniProt:the Universal Protein Resource (www.uniprot.org), which is required forcytosolic sequestration. Upon oxidative stress, interacts with STK4,which disrupts interaction with YWHAB/14-3-3-beta and leads to nucleartranslocation. The subcellular location of FOXO3A is in the cytoplasm,and cytosol. It translocates to the nucleus upon oxidative stress and inthe absence of survival factors. In the presence of survival factorssuch as IGF-1, FOXO3A is phosphorylated on Thr-32 and Ser-253 byAKT1/PKB. This phosphorylated form then interacts with 14-3-3 proteinsand is retained in the cytoplasm. Survival factor withdrawal inducesdephosphorylation and promotes translocation to the nucleus where thedephosphorylated protein induces transcription of target genes andtriggers apoptosis. Although AKT1/PKB doesn't appear to phosphorylateSer-315 directly, it may activate other kinases that triggerphosphorylation at this residue. FOXO3A is phosphorylated by STK4 onSer-209 upon oxidative stress, which leads to dissociation fromYWHAB/14-3-3-beta and nuclear translocation.

Human longevity is a complex phenotype with multiple determinants Whilenon-genetic factors, including diet, physical activity, health habitsand psychosocial factors are important, up to 50% of the variation inhuman lifespan might be explained by genetic differences.¹⁻⁵ Severalstudies suggest that about 25% of the variation in human lifespan inaverage-lived populations can be explained by genetic factors but inpopulations with larger numbers of exceptional survivors the geneticcontribution to lifespan may be much higher. For example, family studiesof nonagenarians and centenarians show that sibling relative risk, acommon method for assessing potential genetic contribution to a complexphenotype,⁶ is particularly high and grows with increasing age of theproband.⁷⁻¹⁰ However, studies of candidate “longevity-associated” genesin humans, hereafter referred to as “longevity genes,” have generallybeen disappointing. Few replications have been observed acrosspopulations, with the exception of the ApoE gene.³

In contrast, there have been several robust genetic findings in modelorganisms of aging.¹¹⁻¹³ For example, variation in single genes canresult in substantial differences in lifespan in model organisms,particularly with genes that are considered part of the insulin/IGF-1(IIS) signaling pathway.¹⁴⁻¹⁸

Mutations that increase SIR-2 activity or that decrease insulin/IGF-1signaling both increase the lifespan of C. elegans by activating theDAF-16/FOXO protein.^(19,20) In mammalian cells, a Sir2 homolog “SIRT1,”influences several downstream transcription events affecting lifespan,including the cellular response to stress. SIRT1 accomplishes this byregulating the FOXO (Forkhead box transcription) factors, a family ofproteins that function as sensors in the IIS pathway and are alsoregulators of longevity in several mammals.¹⁷

Genetic knock-out models in mammals (and other species) have alsosupported the HS hypothesis. For example, mice with a fat-specificinsulin receptor knockout (FIRKO) have reduced fat mass, protectionagainst age-related obesity and have extended longevity.²¹ Many othermutations in the HS pathway appear to impact longevity in mice. Theseinclude mutations in the IGF-1 receptor,²² IRS-1,²² IRS-2,²³ PAPP-A,²⁴and the Ames Dwarf mouse mutation.²²

The basic molecular pathway of insulin signaling is conserved throughevolution, evidence of which can be seen in yeast, flies, worms, rodentsand humans.²⁵ A key regulator of this pathway in worms is thetranscription factor DAF-16 (abnormal DAuer Formation-16), which isrequired for the large lifespan extension produced in C. elegans byinhibiting insulin/IGF-1 signaling.¹⁶ A number of factors appear toextend lifespan in C. elegans in a daf-16 dependent manner, such as AMPkinase,²⁶ 14-3-3 proteins,²⁷ the lin-4 microRNA,²⁸ and heat shockfactor.²⁹ Homologues of DAF-16 in several species have been linked toaging phenotypes and longevity.³⁰ For example, the stress responsiveSun-N-K terminal Kinase (INK) pathway appears to require FOXO to prolonglifespan in Drosophila ³¹ and when flies over express dFOXO, the DAF-16ortholog, it can markedly increase lifespan.³² The remarkableconvergence of such a diverse array of signals on DAF-16/FOXO suggeststhat this protein may be an important, evolutionarily conserved “node”in a signaling network that impacts aging and longevity.

The human homologue of DAF-16 includes four FOXOs: FOXO1, FOXO3A, FOXO4and FOXO6. Therefore, it is tempting to hypothesize that common, naturalvariation in the form of single nucleotide polymorphisms (SNPs) in FOXOand related genes might influence human longevity. “FOXO3” is synonymouswith “FOXO3A” since FOXO3B is a pseudo-gene on chromosome 17.

This is an appealing hypothesis. A connection between insulin, FOXO,oxidative stress and human longevity would be particularly interestingsince oxidative stress has long been a favorite putative mechanism ofaging. Since 1956, the free radical theory of aging has hypothesizedthat aging results partly from damage to DNA, cells and tissues fromcumulative exposure to reactive oxygen molecules³³ and although not yetuniversally accepted, supportive evidence has accumulated over theyears.^(34,35) Thus, FOXO may provide a potential branch-point or bridgebetween insulin signaling, free radicals and human aging/longevity.

There has been some prior work linking genes in the IIS pathway to humanlongevity,^(36,37) including an interesting recent report by Suh etal,³⁸ which links functionally significant IGF-1 receptor mutations toexceptional longevity, but we have not found any published reports ofassociation between FOXO genes and human longevity. Prior studies havefound links between FOXO genes and other aging phenotypes, including4-year survival and stroke risk³⁹ as well as premature menopause.⁴⁰

Human longevity, however, is a complex phenotype that encompassesdisease-specific risks as well as the individual rate of aging. Thestudy of its genetic antecedents is challenging. The study of longevitymay be affected by small genetic effect sizes, population stratificationartifact, population heterogeneity, lack of sufficient numbers oflong-lived study participants, and other problems.^(3,4,41) Therefore,in order to assess potential genetic contributions to human longevityfrom genes linked to IIS signaling, we chose a large, homogeneous,long-lived population of men well characterized for aging phenotypes andwe performed a nested-case control study of 5 candidate longevity geneswith links to the IIS pathway. These genes were chosen based on priorassociations with aging phenotypes principally from gene knockout,transgenic, mutant and other model organisms of aging.^(3,4,14-17,36,42)Priority was given to genes that are involved in insulin sensitivity andglucose (energy) homeostasis.

The rapid aging of the population will place unprecedented challenges onsociety due to increased prevalence of chronic disease and disability.⁴⁵Better understanding of mechanisms of aging, including biologicalpathways that may have widespread influence on how we age, could haveimportant implications for lowering our risk for age-related disease anddisability. There are many biologically plausible candidate genes forhuman longevity but only one finding has so far been widely replicatedin multiple populations, that of the ApoE gene.³ This gene haswidespread effects on aging phenotypes, particularly cardiovasculardisease and dementia, and as such influences the ability to achieve along and healthy life.

SUMMARY OF THE INVENTION

The challenge in finding genes that have widespread effects on humanaging phenotypes and longevity suggests that it may be helpful to usemodel organisms to identify a priori potential candidates beforeconducting human studies. Therefore, we chose to study several candidategenes within the human insulin/IGF-1 signaling pathway and/or oxidativestress response system on the basis of sequence and/or functionalhomology with model organisms of aging or prior human studies. Weconstructed a list of human candidate genes from these signalingpathways and assessed variations in these candidate genes occurring at afrequency of approximately 10% or greater in the Japanese population.Due to limited resources, only three SNPs were chosen from each gene foranalysis. SNPs were selected from regions with linkage disequilibrium(LD), when possible, in order to provide maximal coverage of each gene.

In general the invention provides compositions and methods for detectingthe FOXO3A “GCC” haplotype (e.g. a FOXO3A haplotype associated with anincreased longevity, defined herein as the likelihood of a human subjectliving an additional 15 or more years). In preferred embodiments, thedetected FOXO3A haplotypes are associated with either an increasedlikelihood or a decreased likelihood of living longer, however theinvention necessarily encompasses materials and methods for detecting aFOXO3A haplotype associated with neither an increased nor a decreasedlikelihood of living longer and/or minimizing risk for age-associateddiseases (e.g. a “normal” or “wt” genotype). Age-associated diseasesrefers to coronary heart disease (CHD), also known as coronary arterydiseases, stroke, cancer, chronic obstructive pulmonary disease (COPD)or other chronic lung disease, Parkinson's disease, diabetes, obesity,dementia (and general cognitive function), frailty (ability to walk) orother age -related disease or physical and or cognitive impairment.There may also be an association with obesity in humans.

The “GCC” haplotype encompasses tens of kilobases of DNA. Other SNPs inthis region demonstrate linkage disequilibrium with the three SNPsdescribed herein. It is anticipated that additional SNPs will beidentified within this GCC haplotype that also have an association withlongevity and healthy aging, and may be useful of predictingage-associated diseases. The “GCC haplotype” can serve as a surrogatefor other types of alteration of DNA, either within or adjacent to theFOXO3A gene, that is ultimately found to be the “functional variant”that leads to the prediction of exceptional longevity and/or healthyaging. These other alterations may be in the form of inversions,duplications, deletions, and may include other genes or transcripts thatwere previously unknown, for example, the gene “LOC100130966”.LOC100130966 is similar to SMT3 suppressor of mif two 3 homolog 2 hasbeen identified to lie within exon 2 of the FOXO3A gene, which is withinthe “GCC” haplotype. The DNA sequence for LOC100130966 is described bythe GenBank accession ID# “XM_(—)001725519” and the predicted amino acidsequence of LOC100130966 is described by GenBank file “XP_(—)001725571”.

Haplotype analysis may be used to potentially predict which patientswould benefit by aggressive wellness or disease prevention/treatmentinterventions. Haplotype analysis may be provided in a kit form. Riskcalculators could use such information for purposes of assessinglikelihood of disease, disability or death or determining how many yearsof survival or disease-free survival a person has. Such informationwould be important to patients, health insurance companies, long termcare insurance companies and physicians or other health care providersin order to provide some guidance as to the patient's long-term needs.Pharmaceuticals could be developed that modify the action of the FOXO3Agene, modify the cellular location of the FOXO3A protein and/or itsinteractions with other proteins, or modify the amount or type ofprotein produced by the gene in order to impact health or diseasesrelated to aging.

Homologous sequences in mice may be associated with premature ovarianfailure. Castrillon D H, Miao L, Kollipara R, Horner J W, DePinho R A.Suppression of ovarian follicle activation in mice by the transcriptionfactor Foxo3a. Science. 2003 Jul. 11; 301(5630):215-8. Consequently,similar haplotype analysis can be useful in veterinary applications.

Further features of the invention will now become apparent from thefollowing description, by way of example only, with reference to theaccompanying Figures and Tables.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of an ARMS-PCR assay to detect the FOXO3A G/Tvariants using the primers and conditions described in Table 10.

FIG. 2 illustrates the schematic outline of the ARMS-PCR assay to detectthe FOXO3A G/T variants using the primers described in Table 10.

The following tables are part of the description:

Table 1. Baseline Characteristics of the HHP/HAAS Cohort in 1991-93(n=3,741)

Table 2. Baseline Characteristics by Case-Control Status Table 3.Candidate Genes for Human Longevity and the MAF in Cases and ControlsTable 4. FOXO 3A3 Genotype by Case-Control Status

Table 5. Difference in Health Status between Genotype Groups at Baseline

Table 6. Insulin Sensitivity Phenotypes According to FOXO3A GenotypeTable 7. Prevalence of Aging-related Phenotypes in Relation to FOXO3A3Genotype Table 8. Genotype Distribution by Maximum Attained Age

Table 9. Primers for Identification of the rs2802292 G-T PolymorphismTable 10. PCR Conditions for Identification of rs2802292 G-TPolymorphism

DETAILED DESCRIPTION OF THE INVENTION A. Hawaii Lifespan Study StudyPopulation

This nested-case control study was conducted as part of the HawaiiLifespan Study, an embedded cohort study of healthy aging drawn from theoriginal population of the Honolulu Heart Program (HHP) and HonoluluAsia Aging Study (HAAS). The HHP is a population-based, prospectivestudy of cardiovascular disease among 8,006 Japanese American men thatbegan in 1965. The HHP participants were recruited from 9,877 men withvalid contact information who were born between 1900 and 1919 and livedon the island of Oahu in 1965.⁶¹

Study participants had both parents from Japan, usually the west andsouthern parts of Japan (94% from the central region or further west andsouth); 49% of them had parental origins from the adjacent prefecturesof Hiroshima and Yamaguchi.^(61,62) Although the most participants wereborn in Hawaii (88%), there is a theoretical possibility of confoundingof case control status with allele frequencies due to geographic origin.Therefore, for certain analyses, cases and controls were stratified byparental prefecture of origin using conditional logistic regressionmodels. Analyses showed no evidence for population stratification in thedataset (data not shown).

The HHP cohort recruitment, design, and procedures have been outlined indetail elsewhere.⁶² Briefly, at the time of study enrollment(1965-1968), participants were aged 45 to 68 years (mean age, 54 years).From the commencement of the HHP, information on the development ofincident coronary heart disease and stroke, as well as mortality fromall causes, has been obtained by monitoring obituaries in localnewspapers (English and Japanese) and through surveillance of hospitaldischarge records.⁶¹ A follow-up survey in the 1991-1993 examinationfound that only 5 men could not be traced for mortality information.⁶³

All participants for the current study were drawn from records of studyparticipants updated to August, 2007. Archived phenotypic data and bloodsamples from Exam 4 of the HHP (1991-1993), which coincided with thecommencement of the Honolulu Asia Aging Study (HAAS), was used as thebaseline exam for this nested case-control study. The HAAS was begun asan expansion of the HHP for the study of neurodegenerative diseases,cognitive function and other aging phenotypes in elderly persons.⁶⁴Participants included 3,741 men aged 71 to 93 at Exam 4 (mean age77.9±4.7 years), approximately half the number of the original HHP.⁶⁴

For the purposes of the current nested case-control study, “cases”(longevity phenotype) were defined as all HHP participants who hadsurvived to at least the upper 1% of the 1910 U.S. birth cohort specificsurvival (minimum 95 years of age) from the time of recruitment.^(65,66)A total of 213 individuals who had survived to at least 95 years of age,as of August 2007, were studied. 176 of these individuals had died (meandeath age 97.5; SD 2.1; range of 95-106 years) and 37 individuals werestill alive (mean age 98.7, SD 2.1; range 97-106 years).

The controls consisted of 402 individuals from the HHP/HAAS cohort whodied near the mean death age for the 1910 U.S. birth cohort specificsurvival for middle-aged men (approximately 77 years of age). In orderto achieve a case:control ratio of approximately 1:2, we sampled theHHP/HAAS study population for controls who died up to the age of 81years. The mean age at death for our control population was 78.5 years(SD 1.8, range 73-81 years). This is slightly higher that of the U.S.male population, but consistent with the high average life expectancy ofJapanese-American men in Hawaii, which was 3.5 years longer than whitemales at last report.⁶⁷ All cases and controls were ethnic Japanesewhose families came mainly from Central-West Japan.^(61,62)

Procedures were performed according to institutional guidelines andapproved by the Institutional Review Board of Kuakini Medical Center.Written informed consent was obtained from all study participants orfrom family representatives, if participants could not provide consent.

Genotyping

We chose three SNPs from each of five candidate genes. We chose genesthat have well-described influences on aging pathways in modelorganisms. All genes were chosen based on hypothetical links to the HSpathway and potential links to energy homeostasis, glucose and/or lipidmetabolism, see FIG. 1. SNPs were chosen based on their minor allelefrequencies reported in the HapMap or JSNP database(snp.ims.u-tokyo.ac.jp).

Total cellular DNA was isolated using the PureGene system (GentraSystems, Inc.) quantified using PicoGreen staining (Molecular Probes,Eugene, Oreg.) and SNPs from candidate genes genotyped using allelicdiscrimination assays. Taq Man® (Applied Biosystems, Inc.) reagents werepurchased from ABI and SNPs were chosen with a frequency ≧˜0.1 in theJapanese population (http://www.ncbi.nlm.nih.gov/projects/SNP/). PCR wasamplified under standard conditions using Taq Gold (Perkin-Elmer, Corp)and detection of PCR products with Taq Man® assay, using a 6-FAM-labeledFRET probe for one allele and a VIC-labeled probe for the other allele,using minor groove binding (MGB) quenchers to enhance detection ofassays. PCR products were measured with the ABI Prism 7000 SequenceDetection System.

Genotype data were managed through an integrated database system (MSExcel, Microsoft, Inc). All positive controls on each genotyping platewere also evaluated for consistency. Positive markers were tested fordeviation from Hardy-Weinberg equilibrium. Call rates exceeded 98%.

Statistical Analysis

SNPs were evaluated for deviation from Hardy-Weinberg equilibrium. ThePearson chi-square test was used to compare the cases and controls forequal genotype frequencies using the software program StatXact.⁶⁸ Forestimates of strength of association, odds ratios were estimated usinglogistic regression models from SAS.⁶⁹ General linear model (GLM) andanalysis of covariance (ANCOVA) were further used to compare proportionof healthy study participants by FOXO3A genotype. For the analysis ofaging phenotypes in case and controls, Student's t test for comparingdistribution of continuous variables and Chi square for proportionalvariables.

Results

The baseline characteristics of the HHP/HAAS study population at the1991-1993 examination are presented in Table 1. The mean age was 77.9years and 100% of the population was male and of Japanese ethnicity.Biological characteristics, general health status, disease prevalenceand functional status are presented.

TABLE 1 Baseline Characteristics of the HHP/HAAS Cohort in 1991-93 (n =3,741) Variables at Baseline Exam (1991-93) Mean ± SD Min-Max BiologicalCharacteristics Age (y) 77.88 ± 4.69 71 93 Body Mass Index (BMI) kg/m223.43 ± 3.16 12.25-39.34  Waist/Hip Ratio  0.94 ± 0.06 0.73-1.27  TotalCholesterol (mg/dl)* 189.73 ± 33.16 81-382 Triglyceride level (mg/dl)*148.96 ± 93.85  32-1369 HDL (mg/dl)*  50.94 ± 13.36 20-129 Glucose(mg/dl)* 113.03 ± 29.4  44-399 Insulin (mIU/L)*  16.82 ± 32.48  1.5-1164General Health Status Self-Reported “Poor” Health (%) 32.88 — DiseasePrevalence CHD (%) 19.38 — Stroke (%)  4.73 — Cancer (%) 13.84 —Diabetes (%) 58.11 — Physical and Cognitive Function Lower Body(Difficulty Walking) (%) 20.47 — Upper Body (Grip Strength in kg) 30.09± 6.88 0 55 Cognitive Score (CASI)  82.24 ± 16.37  0-100 *Fasting values

From this 1991-93 baseline population, we selected all participants who,by 2007, had survived to age 95 years or more as “longevity” cases(n=213). We then selected all participants who died before the age of 81years as “average” lived controls (n=402). Baseline characteristics ofthe cases and controls are presented in Table 2. In terms of biologicalcharacteristics, the long-lived cases were older, leaner (lowerwaist:hip ratio), had lower triglycerides (borderline), lower glucose,lower insulin levels and higher prevalence of the FOXO3A3 allele at thebaseline exam. The cases also had better self rated health and lowerprevalence of cardiovascular disease (CHD and stroke) and cancer.Functionally they appeared better able to walk but had lower gripstrength. There was no significant difference in cognitive score.

TABLE 2 Baseline Characteristics by Case-Control Status Average LivedPhenotype Longevity Phenotype Variables at Baseline (Mean Attained Age78.5 y) (Mean Attained Age 97.9 y)* Examination (1991-93) Mean ± SDMin-Max Mean ± SD Min-Max P† Biological‡ Age at Baseline Exam (y) 74.63± 2.05  71-79 85.62 ± 3.12  80-93 <.0001 Body Mass Index (BMI) kg/m²23.4 ± 3.17 15.89-32.33 23.0 ± 2.91 15.4-31.1 0.1272 Waist/Hip Ratio0.95 ± 0.06 0.78-1.15 0.93 ± 0.06 0.73-1.07 0.0008 Total Cholesterol(mg/dl) 187.96 ± 34.6   98-303 185.36 ± 32.16   95-304 0.3680 HDL(mg/dl) 50.82 ± 14.17  21-129 51.29 ± 13.54  27-100 0.6911 Triglycerides(mg/dl) 154.72 ± 118.72  46-1369 140.32 ± 82.23   38-649 0.1178 LogTriglycerides§ 4.88 ± 0.51 3.83-7.22 4.81 ± 0.50 3.64-6.48 0.0965Glucose (mg/dl) 117.83 ± 35.9   69-323 108.98 ± 22.55   77-298 0.0012Insulin (mIU/L) 25.54 ± 82.89   3.3-1164  13.8 ± 11.39  1.5-104 0.0421Log Insulin§ 2.69 ± 0.74 1.19-7.06 2.44 ± 0.58 0.41-4.64 <0.0001 FOXO3A3MAP (rs2802292) 0.255 — 0.371 — <0.0001 General Health Status Self-rated“Poor” Health (%) 41.92 — 31.07 — 0.0163 Disease Prevalence CHD (%)26.37 — 7.55 — <0.0001 Stroke (%) 7.46 — 3.3 — 0.0394 Cancer (%) 20.15 —13.68 — 0.0468 Diabetes (%) 60.55 — 59.81 — 0.8587 Physical/CognitiveFunction Lower Body (Difficulty Walk 30.59 — 16.83 — 0.0002 Upper Body(Grip Strength in 29.85 ± 7.54   0-47 26.37 ± 5.53   8-45 <0.0001Cognitive Score (CASI)¶ 80.96 ± 19.48  0-100 78.54 ± 13.85 12-98 0.1088*Cases (longevity phenotype) consisted of all HHP/HAAS participants withDNA samples (living and dead) who had reached the age of 95 years byAugust 2007: Gp 1: Alive, n = 37, mean age 98.7, range 97-106 years; Gp2: Dead, n = 166, mean death age 97.5, range 95-106 y); †p value fromStudents t test for continuous variables and Chi Square for categoricalvariables; ‡Fasting values; §Log transformation performed for variablesnot normally distributed; ¶CASI (Cognitive Abilities ScreeningInstrument)⁴³

Five genes were investigated (ADIPOQ, FOXO1A, FOXO3A, SIRT1, and COQ7).Minor allele frequencies and other related genetic information for thecases and controls are presented in the Table 3. However, only FOXO3Agenotype was associated with longevity using an initial cut-off value ofp<0.05.

TABLE 3 Candidate Genes for Human Longevity and the MAF in Cases andControls Minor allele freq. (MAF) Gene Name Symbol SNP ID# Variable NameCases Controls P* †Adipo, C1Q, CDC ADIPOQ rs1063539 ADIPOQ_1 0.297 0.2630.20 rs182052 ADIPOQ_2 0.455 0.493 0.22 rs266729 ADIPOQ_3 0.195 0.2390.08 Forkhead Box O1A FOXO1A rs2755209 FOXO1A1 0.272 0.291 0.48rs2721069 FOXO1A2 0.293 0.307 0.62 rs2755213 FOXO1A3 0.350 0.358 0.77Forkhead Box O3A FOXO3A rs2764264 FOXO3A1 0.347 0.248 0.0002 rs13217795FOXO3A2 0.340 0.248 0.0006 rs2802292 FOXO3A3 0.371 0.255 <0.0001 Sirtuin1 SIRT1 rs7069102 SIRT1_1 0.185 0.181 0.84 rs10823112 SIRT1_2 0.3370.360 0.44 rs1885472 SIRT1_3 0.188 0.179 0.71 Coenzyme Q7 COQ7 rs8051232COQ7_1 0.147 0.150 0.90 rs11074359 COQ7_2 0.153 0.171 0.43 rs7192898COQ7_3 0.162 0.170 0.73 *Comparing MAF between cases and controls withChi-square test; †Adipocyte, C1Q, and Collagen Domain Containing.

Variant “rs2764264” has previously been referred to as “rs12524491”. TheDNA Sequence of SNP rs2764264 (“FOXO3A1”) identified as SEQ ID No. 2 is:

TATTTCACTGGCCAGGACCTCCAATACATTGTTGAATAGCAGTGGTGAAAGCAGAGATCCTTACCATTTTTCTCATCTTAAGGGGAAAGCATTCAGTCTTTCACTGTTAAGTATCATGTTAGGTGTAAGTTTGTCACATATTTCCTTTATCAGGCTGAGGTAGTTTTCTCTATTCCTATGTGTTGAGTAGTTTTTGTTTTTTAAATTATGAGTGGATATTGAATTTTGTCAGATGCTTTTTCCTCACCTGTTGAGAAGATCAGATGGTTTTTCTTTTTCAGTCTTTTAATATGATGAAATACATTGACTGATTTGCAATGTTAAACCAACCTTACATTCCTGGGATAAATCCCACCTGGTCTTGATATGTTACCATGAGATTCAAGTAGCTAAAATTTTGTTAAGGATTTTTGTGTCTGTCTTCATGAGGAATATTGATCTATACATTTCTTATAATATCTTTGCCTGTTTTTGGTACCAGGGTAATGGTGGTCTTATAA (C/T)ATGAGTTGGA AAGTGTTCCC TGTTCTGCTC TGGTAGCACT GTAGTATCTCTTCCTTAAATGTTTGGTAGAATTCAACGGCAGTTAAGCCATCAGAGCCTGGAGTTTTTTTGTGTGTGAGGAAATGTTTAACTGCTAATTCAATTTATTTCATAGATACAATGCTGTTGGCTTGTCTGTTTCTTCTTGAATGAGTTTTGGTAGTCTGTGTCTTTTAAGGAATTTGCCCATTTTATTTAAGTTGTCTAATTTATGGGCATAAAGTCATTTATAATGTTCTCTTATTATCCTTTTAATAGATATATCATCTGTAGTGATTTCATTTTCATTCCTGATGTTGATAATTTGTCTTAACTCCCTTTCCCCCTCATTCCTTATCTGTTTAGTGCCTTGCAATTTCATTGATCTTTTAAACGAATTAACATTTGCTTCCACTGACTTTTCCCCCGTTACTTTTATGTTTTTACTTCCATTGATTTTTTTTTTCTCTTTTAATCTTTTA

The DNA Sequence of SNP rs13217795 (“FOXO3A2”) is identified as SEQ IDNo. 3 is:

CACCACCACCCACTAGACAAATTGCTTAACCTTTCTGCACCTCAGTTTCCTCCTGACAGGCTTGTTTAGAAAATAAAATGAGATCAAATTTGTCAAGCACAGAGCATTGGCCCTGGTAGGCACCACATACATGAATTTCCTTCAGATTGTAGGTGAAGTAGACTTGATTTGGGATTTCTCTTGTTACCTAGGTGCTTGTGTAGAGGAGACTTTAGAACCAGAATGTGTTATTTGTGGTTTTGAGTGTGCCTGGGACTCTGAGCCAACTGAATTACCAAGTAATGGGGGCCCCATGGCATC (C/T)CATGACAGGTGGAGAGCCGGCTCTTCACCCTGGATGGACCTGAAATGCCTGCTAAGGCCTTGCTCCACCGAGTAGCACACACCCTATCAGTTTGCCCTTCTTTCCATCTCTTATTCTAGAGACCTTAAAGCCTACTTGTTGGTATATATTTTCAGGTTTTTGGAAATTGGGCTGTTTAATTGAAGTTAATACCAGTGATGAGACTTTTCAACCTGAGAACAACCTAGATGCTACTTCACATTTTGCAGTGGAAGCTTACTTCCATCTTCACTCATGTAGGACATTCTTTGGTCTCAATGT

The DNA Sequence of SNP rs2802292 (“FOXO3A3”) identified as SEQ ID No. 4is:

TGAAGCAGGGCATCAGGGAATGGGAGTTGGTGAGGAAATTACATTAACATTTATTGAGCACCATTCTCACTATAAACCTGAACGTAAATATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTATTTTAGTAGAGATGAGGCCTTGCTGTGTTGCCTAGACTGGTCTTGAACTCCTGGGCTCAAGCAATCCTCTCACCTTGGCCTCTCAAAGTGCCTCTCAAAGGTGTGAGCCACCATGCCCAGCCTATTCGTTTTTAATTTCTGAAGAAACTGAGGCTAACAGCTGGGTCTGGCCCATGACTGGTTCAGTTGGTATTTGGTGGACCAAGTTGACCAAGCTCACCCAGCTTCTGAGTGACAGAGTGAATATAAACCCAGCCTGCTCACTCCATTTCCTAGTTTTCTCACCTCTACCAGGGTCTCTGTTGCTCACAA GAGCTCAGGGCTGGGA(G/T) AAGCCTCTGTGTGACAGATGAAGGGGTCCTGCTGCTCTCTAGGGAAGAATCGGTCCCAAATTGCTCAAGGGAGTAAGGTGGTTTCGTTGAGGAGCATCAGCTAGGGGGATTGATGGGAATAGGTGTCAGGCAGCCAGTGGAAATTTTGTGTGCCCACCTGTGGCACATGTATTATGCAAATTCATGCAAAAATATATATA

The “GCC haplotype” can be described using SNPs rs2764264, rs13217795,and rs2802292 and is the allele that contains the following combinationof genotypes:

rs2764264 rs13217795 rs2802292 “C” “C” “G”

When viewing these variants from the top of the chromosome (lowernucleotide position on the genetic map) to the bottom (higher nucleotideposition on the genetic map) the “GCC haplotype” can be described usingSNPs rs2802292, rs2764264, and rs13217795 according to NCBInomenclature, and is the allele that contains the following combinationof genotypes:

SNP ID # rs2802292 rs2764264 rs13217795 SNP Variable Name from FOXO3A3FOXO3A1 FOXO3A2 Table 3 Chromosome 6 Nucleotide 109015211 109041154109080791 Position SNP Allele “G” “C” “C”

The above data are from:

Database of Single Nucleotide Polymorphisms (dbSNP). Bethesda (Md.):National Center for Biotechnology Information, National Library ofMedicine. (dbSNP Build ID: 129, NCBI genome build 36.3). Available from:http://www.ncbi.nlm.nih.gov/SNP/⁷⁰

Further investigation comparing the genotype frequencies of FOXO3A3between cases and controls revealed a highly significant difference withan exact p value of 0.00009 using the permutation distribution of thePearson's chi-square statistic. These results are presented in Table 4.There were five loci with 3 SNPs within each allele in this study (Table3) so Bonferroni adjustment for multiple comparisons resulted in acorrected p value of 15×0.00009=0.00135. Due to the high link LD betweenthe 3 SNPs of FOXO3A, we further investigated the FOXO3A3 SNP only(rs2802292). The odds ratio (OR) for homozygous minor vs. homozygousmajor alleles for FOXO3A3 between the cases and controls was 2.75 (95%CI: 1.51−5.02, p=0.0007), and the OR for heterozygous vs. homozygousmajor alleles between the cases and controls was 1.91 (95% CI:1.34−2.72, p=0.0003). These results suggest an additive effect onlongevity.

TABLE 4 FOXO 3A3 Genotype by Case-Control Status FOXO 3A3 Genotype (rs2802292) Case-Control Status TT TG GG Average-Lived Phenotype* 223 (55%)153 (38%) 26 (6%)  Longevity Phenotype†  81 (38%) 106 (50%) 26 (12%) pvalue for Pearson Exact test‡ 0.000091 p value after Bonferroniadjustment 0.00135 *number and % of subjects from n = 402“average-lived” decreased controls (mean attained age 78.5 years)†number and percent of subjects from n = 213 “long-lived” cases (meanattained age 97.9 years) ‡From the exact Pearson Chi-square testcomparing the genotype frequencies in the cases and controls.

In order to understand more about the longevity phenotype at youngerages, we compared the proportion of people who were healthy at thebaseline exam (1991-93) for each of the three FOXO3A genotype groupsusing the definition of healthy survival from Willcox et al. (2006).⁴⁴The differences were highly significant (Table 5). Those who possessedone or more G alleles were much more likely to be healthy at baselinethan those who were homozygous for the major (TT) allele. Approximately75% of those homozygous for the minor allele were healthy at thebaseline exam versus only about 57% of those homozygous for the majorallele. After adjusting for case-control status, the differences werestill marginally significant. This suggests that there was remainingassociation of the allele with health status within the categories oflong term survivors (cases) and controls.

TABLE 5 Difference in Health Status between Genotype Groups at Baseline% Healthy at Baseline* p for trend Homo. Homo. Un- Adj. for Case- MajorHeter. Minor adjusted Control Slat FOXO3A1 57.41 69.48 75.51 0.01 0.065FOXO3A2 57.37 69.35 77.08 0.01 0.035 FOXO3A3 57.89 68.34 75.00 0.020.097 *“Healthy” defined as absence of six major chronic diseases (CHD;stroke, cancer, PD, COPD and treated Type 2 DM; high physical function(can walk ½ mile) and high cognitive function (CASI score >74).

In order to assess whether there was a relation between insulinsensitivity, a potential intermediate phenotype of longevity, andgenotype, we tested the relation between fasting insulin, glucose, HOMAand genotype (Table 6). For non-normally distributed variables we usedlog conversion to a normal distribution. There was a significantrelation between insulin, log insulin, HOMA and genotype. Homozygosityfor the G allele was associated with markedly lower insulin, log insulinand HOMA score, but in controls only.

TABLE 6 Insulin Sensitivity Phenotypes According to FOXO3A GenotypeFOXO3A Genotype (rs 2802292) TT TG GG P* Fasting Glucose (mg/dl)Average-Lived 118.4 ± 34.0  117.4 ± 38.0  115.9 ± 40.1 0.80 Long-Lived108.3 ± 20.7  109.1 ± 23.7  110.5 ± 24.1 0.73 Fasting Insulin (mIU/L)Average-Lived 23.7 ± 81.2 30.4 ± 91.9 13.2 ± 5.9 0.004 Long-Lived 13.5 ±9.0  14.1 ± 13.4 13.3 ± 9.3 0.77 Log Fasting Insulin (mIU/L)Average-Lived 2.68 ± 0.67 2.73 ± 0.85  2.47 ± 0.48 0.03 Long-Lived 2.45± 0.55 2.43 ± 0.61  2.44 ± 0.52 0.99 HOMA IR Score Average-Lived  9.1 ±53.0 10.0 ± 32.2  3.8 ± 2.4 0.03 Long-Lived 3.7 ± 2.8 4.0 ± 4.3  3.6 ±2.2 0.55 *p-value for Student's t-test comparing mean values between GGgenotype and other groups within cases and controls.

We also tested for a relation between lifetime prevalence of severalchronic diseases and FOXO3A genotype in study participants (Table 7).

TABLE 7 Prevalence of Aging-related Phenotypes in Relation to FOXO3A3Genotype FOXO3A3 Genotype TT TG GG p CHD prevalence (%) Average-Lived32.3 18.3 23.1 0.010 Long-Lived  7.4  7.6  7.7 0.998 All 25.7 14.0 15.40.002 Stroke prevalence (%) Average-Lived  6.7  8.5  7.7 0.813Long-Lived  4.9  1.9  3.8 0.510 All  6.3  5.8  5.8 0.974 Cancerprevalence (%) Average-Lived 22.4 18.3 11.5 0.326 Long-Lived 17.3 12.4 7.7 0.400 All 21.1 15.9  9.6 0.075 Diabetes prevalence (%)Average-Lived 60.6 62.3 50.0 0.498 Long-Lived 57.5 64.1 50.0 0.368 All59.8 63.0 50.0 0.212 p value based on Chi-Square test comparingfrequency of GG genotype to other genotypes for average lived controls(n = 402), long lived cases (n = 213) and all subjects (n = 615).

A significant protective relation was found for homozygosity for the Gallele with regard to prevalence of CHD and a borderline relation forcancer and cognitive function. Finally, we assessed the FOXO3A3 minorallele frequency (MAF) distribution by maximum attained age in allparticipants combined (cases and controls). The MAF increased markedlywith age, as expected by earlier case-control analyses (Table 8).

TABLE 8 Genotype Distribution by Maximum Attained Age MAF of Age atDeath (years)* N FOXO3A3 72-74 17 0.21 75-79 277 0.25 80-81 108 0.2695-99 185 0.37 100-106 28 0.39 *37 “long-lived” cases were still alive;mean age of 98.7 y (range 97-106).

The analysis of five candidate genes demonstrated that one gene clearlystood out from the others in terms of a potential human longevity geneFOXO3A. That this gene might be important to human longevity issupported by several lines of evidence. First, in nested case-controlanalyses, variation within this gene was strongly associated withlongevity. The odds ratio (OR) for being homozygous minor vs. homozygousmajor for FOXO3A3 allele (rs 2802292) between the cases and controls was2.75 (95% CI: 1.51−5.02, p=0.0007), and the OR for heterozygous vs.homozygous major between the cases and controls was 1.91 (95% CI:1.34−2.72, p=0.0003). These results suggest an additive effect of theFOXO3A3 G allele on longevity. (i.e., two copies of the G alleleconferred about twice the protective effect). Consistent with this, theminor allele frequency rose markedly with age of the study participants,from septuagenarian to centenarian ages (Table 8).

Second, all three SNPs that were assessed in the FOXO3A gene, which werein tight linkage disequilibrium (LD), were strongly correlated with thelongevity phenotype. This indicates that the finding was unlikely due tochance. Third, those who possessed one or more of the minor (G) alleleswere much more likely to be healthy at the baseline exam, approximately15 years prior, than those homozygous for the major (TT) allele. About75% of those homozygous for the minor allele were healthy at baselineexam versus only about 57% of those homozygous for the major (TT) allele(Table 5).

In fact, the baseline exam suggested that cases were markedly healthierthan controls despite the fact that cases were, on average, 11 yearsolder. The cases possessed significantly less age-related disease,including less prevalent CHD, stroke, and cancer. They also had betterself-rated health and generally had high physical function, includingless difficulty walking. Interestingly, despite being more than a decadeolder than controls, the longevity cases had similar levels of cognitivefunction. This supports the existence of a “healthy aging” phenotypewhere individuals somehow delay or avoid major clinical disease anddisability until late in life. The healthy aging phenotype that weobserved in cases is similar to the healthy aging phenotypes reported incentenarians at younger ages when compared to their age-matched birthcohorts⁴⁶⁻⁴⁸ and in centenarian offspring.⁴⁹ Long-lived cases also hadmetabolic profiles that suggested higher insulin sensitivity at youngerages, with lower waist to hip ratio, lower glucose levels, lower insulinlevels and lower HOMA values (Tables 2 and 6). Several phenotypes wereassociated with variation in FOXO3A genotype.

Surprisingly, there was no significant difference in diabetes prevalencebetween cases and controls. However, since the cases were more than adecade older than controls, and diabetes tends to increase markedly withage, it is noteworthy that prevalence of diabetes was not significantlydifferent. In fact, both cases and controls had a high prevalence ofdiabetes (near 60%), despite relatively low BMI. Why Type 2 diabetestends to be more prevalent in Japanese at a relatively low BMI is notcompletely understood.⁵⁰ However, there may be metabolic differences inJapanese (and some other Asians) with higher visceral fat in Asians atlower BMI than whites and blacks.^(51,52) Indeed, Japan nationalguidelines reflect such population differences and consider Japaneseobese at a BMI of 25.⁵³ Other contributing factors to the highprevalence of diabetes in the HHP/HAAS cohort include the fact that allparticipants were tested for diabetes by several different clinicaltests and at several prior examinations making detection more likely.

Of note, FOXO3A genotype was significantly associated with plasmainsulin levels as well as CHD, cancer and Type 2 diabetes prevalence.This is consistent with a known role for FOXO as a mediator of theeffects of insulin and insulin-like growth factors on diversephysiological functions, including cell proliferation, apoptosis andmetabolism.^(17,54) Genetic studies in C. elegans and Drosophila haveshown that FOXO proteins are ancient targets of insulin-like signalingthat regulate metabolism and longevity. Additional work in mammaliancells has shown that FOXO proteins are the targets of protein kinases,influence cell cycle progression, and regulate resistance to oxidativestress in vitro.⁵⁴ In vivo studies have shown that FOXO modifies hepaticglucose output in response to insulin and mediates other metabolicactions.⁵⁴ This strengthens the evidence that FOXO proteins may mediateinsulin effects on metabolism and influence longevity in humans.

Overall, the totality of the evidence supports a potential role ofFOXO3A in human health, aging and longevity. The association of FOXOwith diverse aging phenotypes, including insulin sensitivity, CHD,cancer, Type 2 diabetes and longevity, is suggestive of a “gatekeeper”role in the IIS pathway. An important downstream mechanism wherebyFOXO3A might influence human aging is through modification of oxidativestress—a long held theory of how we age,³³ although we have no directevidence for this in the current study. However, since FOXO genes arethe closest human homologues of C. elegans DAF-16, which protects cellsfrom oxidative stress, this is a plausible mechanism of action formodification of human aging.¹⁷ In C. elegans, DAF-16 increases theexpression of manganese superoxide dismutase (SOD2), which convertssuperoxide to less damaging hydrogen peroxide and is a potent endogenousprotector against free radicals,⁵⁵ among other “anti-aging” effects. Invivo studies show that oxidative lesions in DNA, proteins and othertissues accumulate with age and feeding calorically restricted diets (apotent insulin sensitizer) to rodents ⁵⁶ and humans⁵⁷ mitigates thisdamage.

While FOXO was clearly associated longevity we did not observe a strongeffect of genotype on insulin sensitivity in cases—just controls.However, the GG genotype demonstrated similarly low plasma insulinlevels in both cases and controls, consistent with a modulating effectof genotype on insulin levels in both groups. It is tempting tospeculate that since the cases showed greater insulin sensitivity nomatter what their genotype that they have multiple mechanisms tomaintain insulin sensitivity other than FOXO. This would be consistentwith the hypothesis that most longevity genes have modest or smalleffect sizes. It is also possible that small sample size limited ourability to detect differences in the cases. On the other hand,long-lived mice carrying mutations in either IRS-1⁵⁸ or IRS-2²³ areactually insulin resistant, so insulin sensitivity is not a necessarycondition for mutations in the IIS pathway to be able to confer greaterlongevity.

However, it is interesting to note that in C. elegans, several genesthat by themselves may have small effects on lifespan, are influenced bythe transcription regulating “master gene” DAF-16.⁵⁹ Small differencesin FOXO3A that may be otherwise difficult to detect, could theoreticallymodify several downstream genes related to DNA binding, protein-proteininteractions, cell cycle progression, apoptosis and metabolism. In thismanner, a small modifying effect by FOXO3A potentially has larger,additive downstream effects on aging phenotypes and longevity.

Supportive evidence is beginning to accumulate for a role ofinsulin-signaling in human aging and longevity but the genes that mightmediate these effects are not known. Prior studies have found over orunder representation of single nucleotide polymorphisms (SNPs) from theinsulin-IGF-1 signaling pathway in long-lived humans of Italian,³⁶Japanese,^(37,42) Dutch and Ashkenazi Jewish³⁸ ethnicity, with links toseveral aging phenotypes. While some of these findings have been limitedby small effect sizes and marginal statistical significance, the studyby Suh et al.³⁸ also demonstrated that functionally significantmutations in the IGF-1 receptor exist in some long-lived humans, such ascentenarians.

To date, there has little study of FOXO genes and phenotypes of aging inhumans. Two recent studies suggest that FOXO genes deserve furtherscrutiny. First, a longitudinal study of elderly Dutch men and womenfound that a FOXO1A haplotype predicted 4-year survival and that aFOXO3A haplotype predicted stroke risk.³⁹ Second, the Framingham Study,in a genome-wide association analysis, found that a FOXO3A SNP wasstrongly associated with age at natural menopause in women (p=0.00003).However, the Dutch findings were not statistically significant whenaccounting for multiple comparisons and both studies need replication.The present study is supportive and extends the associations of FOXO3Ato human longevity and insulin sensitivity.

One of the major advantages of the current study is that it employed anested case-control design. This study design selects cases and controlsfrom an ongoing cohort study with longitudinally collected data.Therefore, several phenotypes of interest (e.g. disease prevalence,health status, function) were obtained by direct clinical examinationwhen the participants were younger making the data less subject torecall bias. Recall bias, where study results are less accurate due todifficulty in remembering past events, can be a significant challengewith older adults.

Indeed, studies of exceptional survivors, such as centenarians, thathave found evidence for phenotypes suggestive of slower aging⁴⁶⁻⁴⁸ couldpotentially suffer from significant recall bias. That is, olderparticipants may not recall precisely their past medical history andtheir past functional status. However, in the current study, majordiseases were adjudicated by a morbidity and mortality committee andperformance-based measures of physical and cognitive function were usedto supplement self-reports, and evidence was found for such a healthyaging phenotype. This lends prospective support to previousretrospective work.

There are several other strengths to this study. First, the candidategenes selected for analysis were chosen a priori based onhypothesis-driven criteria. That is, studies of models organisms ofaging employing various methods, particularly knockouts, have shown thatthe IIS pathway is important for aging and longevity. And many functionsappear to be evolutionarily conserved. Second, the findings are strong,highly significant, and include several adjacent SNPs in the FOXO3Agene. Third, the findings are biologically plausible and support theprior findings in animal models of aging and also support the limitedprior human studies. Fourth, the case-control associations withlongevity were detected using a nested case-control analysis with a highevent rate (deaths) during a long period of follow-up. Fifth, the HHPcohort is a highly homogenous cohort and there was no populationstratification detected in our study participants.

A possible drawback is that since the cases and controls had an averageage difference of 11 years we cannot exclude birth cohort as aconfounder. But this is unlikely since there was a maximum 19-yeardifference in birth years between participants. Also, sub analysesrevealed no differences in education and occupation (data not shown)between cases and controls. Moreover, it was the participants who wereolder at baseline who were more likely to have lived to 95-plus yearsand thus obtain the longevity phenotype. Most cohort effects show healthadvantages for younger cohorts. Another possible drawback is that thisstudy was conducted in only one population and thus should be replicatedin other populations in order to assess its generalizability.

In summary, we found that common, natural genetic variation within theFOXO3A gene was strongly associated with human longevity. The prevalenceof the protective allele increased markedly with age. Long-lived caseswere also more likely to possess several additional phenotypes linked tohealthy aging, including lower prevalence of cancer and cardiovasculardisease, better self-reported health, high functional status and theyexhibited several biological markers suggestive of greater insulinsensitivity at the baseline exam. Finally, particular variants withinthe FOXO3A gene were also associated with several of these agingphenotypes, including insulin sensitivity, a putative intermediatephenotype of longevity.

B. Detection of Alleles in Patients (Human and Non-Human)

Many methods are available for detecting specific alleles at polymorphicloci. The preferred method for detecting a specific polymorphic allelewill depend, in part, upon the molecular nature of the polymorphism. Forexample, the various allelic forms of the polymorphic locus may differby a single base-pair of the DNA. Such single nucleotide polymorphisms(or SNPs) are major contributors to genetic variation, comprising some80% of all known polymorphisms, and their density in the human genome isestimated to be on average 1 per 1,000 base pairs. SNPs are mostfrequently biallelic—occurring in only two different forms (although upto four different forms of an SNP, corresponding to the four differentnucleotide bases occurring in DNA, are theoretically possible).Nevertheless, SNPs are mutationally more stable than otherpolymorphisms, making them suitable for association studies in whichlinkage disequilibrium between markers and an unknown variant is used tomap disease-causing mutations. In addition, because SNPs typically haveonly two alleles, they can be genotyped by a simple plus/minus assayrather than a length measurement, making them more amenable toautomation.

A variety of methods are available for detecting the presence of aparticular single nucleotide polymorphic allele in an individual.Advancements in this field have provided accurate, easy, and inexpensivelarge-scale SNP genotyping. Most recently, for example, several newtechniques have been described including dynamic allele-specifichybridization (DASH), microplate array diagonal gel electrophoresis(MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMansystem as well as various DNA “chip” technologies such as the AffymetrixSNP chips. These methods require amplification of the target geneticregion, typically by PCR. Still other newly developed methods, based onthe generation of small signal molecules by invasive cleavage followedby mass spectrometry or immobilized padlock probes and rolling-circleamplification, might eventually eliminate the need for PCR. Several ofthe methods known in the art for detecting specific single nucleotidepolymorphisms are summarized below. The method of the present inventionis understood to include all available methods.

Several methods have been developed to facilitate analysis of singlenucleotide polymorphisms. In one embodiment, the single basepolymorphism can be detected by using a specializedexonuclease-resistant nucleotide, as disclosed, e.g., in Mundy, C. R.(U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide present in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site.Cohen, D. et al. (French Patent 2,650,840; PCT Appln. No. WO91/02087).As in the Mundy method of U.S. Pat. No. 4,656,127, a primer is employedthat is complementary to allelic sequences immediately 3′ to apolymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA™ isdescribed by Goelet, P. et al. (PCT Appln. No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet, P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Recently, several primer-guided nucleotide incorporation procedures forassaying polymorphic sites in DNA have been described (Komher, J. S. etal., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl. AcidsRes. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993). These methods differ from GBA™ in that they all relyon the incorporation of labeled deoxynucleotides to discriminate betweenbases at a polymorphic site. In such a format, since the signal isproportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van derLuijt, et. al., (1994) Genomics 20:1-4). For PTT, RNA is initiallyisolated from available tissue and reverse-transcribed, and the segmentof interest is amplified by PCR. The products of reverse transcriptionPCR are then used as a template for nested PCR amplification with aprimer that contains an RNA polymerase promoter and a sequence forinitiating eukaryotic translation. After amplification of the region ofinterest, the unique motifs incorporated into the primer permitsequential in vitro transcription and translation of the PCR products.Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis oftranslation products, the appearance of truncated polypeptides signalsthe presence of a mutation that causes premature termination oftranslation. In a variation of this technique, DNA (as opposed to RNA)is used as a PCR template when the target region of interest is derivedfrom a single exon.

Any cell type or tissue may be utilized to obtain nucleic acid samplesfor use in the diagnostics described herein. In a preferred embodiment,the DNA sample is obtained from a bodily fluid, e.g. blood, obtained byknown techniques (e.g. venipuncture) or saliva. Alternatively, nucleicacid tests can be performed on dry samples (e.g. hair or skin). Whenusing RNA or protein, the cells or tissues that may be utilized mustexpress an FOXO3A gene.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, N.Y.).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

A preferred detection method is allele specific hybridization usingprobes overlapping a region of at least one allele of an FOXO3Ahaplotype and having about 5, 10, 20, 25, or 30 nucleotides around themutation or polymorphic region. In a preferred embodiment of theinvention, several probes capable of hybridizing specifically to otherallelic variants are attached to a solid phase support, e.g., a “chip”(which can hold up to about 250,000 oligonucleotides). Oligonucleotidescan be bound to a solid support by a variety of processes, includinglithography. Mutation detection analysis using these chips comprisingoligonucleotides, also termed “DNA probe arrays” is described e.g., inCronin et al. (1996) Human Mutation 7:244. In one embodiment, a chipcomprises all the allelic variants of at least one polymorphic region ofa gene. The solid phase support is then contacted with a test nucleicacid and hybridization to the specific probes is detected. Accordingly,the identity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment.

These techniques may also comprise the step of amplifying the nucleicacid before analysis. Amplification techniques are known to those ofskill in the art and include, but are not limited to cloning, polymerasechain reaction (PCR), polymerase chain reaction of specific alleles(ASA), ligase chain reaction (LCR), nested polymerase chain reaction,self sustained sequence replication (Guatelli, J. C. et al., 1990, Proc.Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system(Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), andQ-Beta Replicase (Lizardi, P. M. et al., 1988, Bio/Technology 6:1197).

Amplification products may be assayed in a variety of ways, includingsize analysis, restriction digestion followed by size analysis,detecting specific tagged oligonucleotide primers in the reactionproducts, allele-specific oligonucleotide (ASO) hybridization, allelespecific 5′ exonuclease detection, sequencing, hybridization, and thelike.

PCR based detection means can include multiplex amplification of aplurality of markers simultaneously. For example, it is well known inthe art to select PCR primers to generate PCR products that do notoverlap in size and can be analyzed simultaneously. Alternatively, it ispossible to amplify different markers with primers that aredifferentially labeled and thus can each be differentially detected. Ofcourse, hybridization based detection means allow the differentialdetection of multiple PCR products in a sample. Other techniques areknown in the art to allow multiplex analyses of a plurality of markers.

In a merely illustrative embodiment, the method includes the steps of(i) collecting a sample of cells from a patient (from saliva, cheekswab, blood or other body fluid or component), (ii) isolating nucleicacid (e.g., genomic, mRNA or both) from the cells of the sample, (iii)contacting the nucleic acid sample with one or more primers whichspecifically hybridize 5′ and 3′ to at least one allele of an FOXO3Ahaplotype under conditions such that hybridization and amplification ofthe allele occurs, and (iv) detecting the amplification product. Thesedetection schemes are especially useful for the detection of nucleicacid molecules if such molecules are present in very low numbers.

In a preferred embodiment of the subject assay, the allele of an FOXO3Ahaplotype is identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the allele. Exemplarysequencing reactions include those based on techniques developed byMaxim and Gilbert ((1977) Proc. Natl. Acad Sci USA 74:560) or Sanger(Sanger et al (1977) Proc. Nat. Acad. Sci. USA 74:5463). It is alsocontemplated that any of a variety of automated sequencing proceduresmay be utilized when performing the subject assays (see, for exampleBiotechniques (1995) 19:448), including sequencing by mass spectrometry(see, for example PCT publication WO 94/16101; Cohen et al. (1996) AdvChromatogr 36:127-162; and Griffin et al. (1993) Appl Biochem Biotechnol38:147-159). It will be evident to one of skill in the art that, forcertain embodiments, the occurrence of only one, two or three of thenucleic acid bases need be determined in the sequencing reaction. Forinstance, A-track or the like, e.g., where only one nucleic acid isdetected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labeled) RNA or DNA containing the wild-typeallele with the sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such as whichwill exist due to base pair mismatches between the control and samplestrands. For instance, RNA/DNA duplexes can be treated with RNase andDNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing polyacrylamide gels to determine the site of mutation. See,for example, Cotton et al (1988) Proc. Natl. Acad Sci USA 85:4397; andSaleeba et al (1992) Methods Enzymol. 217:286-295. In a preferredembodiment, the control DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes). For example, the muttenzyme of E. coli cleaves A at G/A mismatches and the thymidine DNAglycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.(1994) Carcinogenesis 15:1657-1662). According to an exemplaryembodiment, a probe based on an allele of an FOXO3A locus haplotype ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike See, for example, U.S. Pat. No. 5,459,039, which is incorporated byreference herein in its entirety.

In other embodiments, alterations in electrophoretic mobility will beused to identify an FOXO3A locus allele. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci. USA 86:2766, see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl9:73-79). Single-stranded DNA fragments of sample and control FOXO3Alocus alleles would be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of alleles in polyacrylamidegels containing a gradient of denaturant is assayed using denaturinggradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature313:495). When DGGE is used as the method of analysis, DNA will bemodified to insure that it does not completely denature, for example byadding a GC clamp of approximately 40 bp of high-melting GC-rich DNA byPCR. In a further embodiment, a temperature gradient is used in place ofa denaturing agent gradient to identify differences in the mobility ofcontrol and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem265:12753).

Examples of other techniques for detecting alleles include, but are notlimited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl. Acad. Sci. USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

Alternatively, the allele specific amplification technology, whichdepends on selective PCR amplification, may be used in conjunction withthe instant invention. Oligonucleotides used as primers for specificamplification may carry the mutation or polymorphic region of interestin the center of the molecule (so that amplification depends ondifferential hybridization) (Gibbs et al (1989) Nucleic Acids Res.17:2437-2448) or at the extreme 3′ end of one primer where, underappropriate conditions, mismatch can prevent, or reduce polymeraseextension (Prossner (1993) Tibtech 11:238. In addition it may bedesirable to introduce a novel restriction site in the region of themutation to create cleavage based detection (Gasparini et al (1992) Mol.Cell. Probes 6:1). It is anticipated that in certain embodimentsamplification may also be performed using Taq ligase for amplification(Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases,ligation will occur only if there is a perfect match at the 3′ end ofthe 5′ sequence making it possible to detect the presence of a knownmutation at a specific site by looking for the presence or absence ofamplification.

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and in Landegren, U. et al. ((1988) Science241:1077-1080). The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al. (1990)Proc. Natl. Acad. Sci. USA 87:8923-27). In this method, PCR is used toachieve the exponential amplification of target DNA, which is thendetected using OLA.

Several techniques based on this OLA method have been developed andcould be used to detect alleles of an FOXO3a locus haplotype. Forexample, U.S. Pat. No. 5,593,826 discloses an OLA using anoligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in To be et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

Another embodiment of the invention is directed to kits for detecting alikelihood for long life or the need for wellness or diagnosticintervention in the near future. This kit may contain one or moreoligonucleotides, including 5′ and 3′ oligonucleotides that hybridize 5′and 3′ to at least one allele of a FOXO3A locus haplotype. PCRamplification oligonucleotides should hybridize between 25 and 2500 basepairs apart, preferably between about 100 and about 500 bases apart, inorder to produce a PCR product of convenient size for subsequentanalysis.

Particularly preferred primers include nucleotide sequences described inSEQ IDs Nos. 2-9. Suitable primers for the detection of a humanpolymorphism in these genes can be readily designed using this sequenceinformation and standard techniques known in the art for the design andoptimization of primers sequences. Optimal design of such primersequences can be achieved, for example, by the use of commerciallyavailable primer selection programs such as Primer 2.1, Primer 3 orGeneFisher.

An example of a simple method for the detection of the “GCC haplotype”involves the use of allele-specific primers that amplify the specificnucleotide of interest, similar to that described in paragraph [0067].This method exploits the fact that oligonucleotide primers must beperfectly annealed at their 3′ ends for a DNA polymerase to extend theseprimers during PCR. By designing oligonucleotide primers that match onlya specific DNA point difference, such as that found in the rs2802292polymorphisms—primers that do not bind the T-type allele—such primerscan distinguish between polymorphic alleles. It is necessary to set up acontrol reaction in the same tube as the amplification refractorymutation system reaction (ARMS) to ensure that lack of productgeneration from a given sample is not simply due to failure of the PCRreaction rather than absence of the “G” variant that the assay isprobing for. Oligonucleotides used for this purpose included forwardouter (“rs2802292_FO”), 5′-GAAACTGAGGCTAACAGCTGGGTCTGGCCC-3′ identifiedas SEQ ID No. 5; reverse outer (“rs2802292_RO”),5′-AGCTGATGCTCCTCAACGAAACCACCTTAC-3′ identified as SEQ ID No. 6; reverseG-specific (“rs2802292_RG”), 5′-GGACCCCTTCATCTGTCACACAGAGGCTcC-3′identified as SEQ ID No.7; and forward T-specific (“rs2802292_FT”),5′-CTGTTGCTCACAAGAGCTCAGGGCTGGGcT-3′ identified as SEQ ID No.8, wherethe underlined final base in the latter two primers anneals at the siteof the G-T difference, whereas the 2nd by from the 3′ end (lowercase) isintentionally mismatched to maximize allelic specificity. The fourprimers in this illustrative example are set forth in Table No. 9.

TABLE 9 Primers for Identification of the rs2802292 G-T PolymporphismPrimer Sequence forward outer 5′-GAAACTGAGGCTAACAGCTGGGTCTGGCCC-3′“rs2802292_FO” reverse outer 5′-AGCTGATGCTCCTCAACGAAACCACCTTAC-3′“rs2802292_RO” reverse G-specific 5′-GGACCCCTTCATCTGTCACACAGAGGCTcC-3′“rs2802292_RG” forward T-specific 5′-CTGTTGCTCACAAGAGCTCAGGGCTGGGcT-3′“rs2802292_FT”(Table 9 discloses SEQ ID NOS 5-8 respectively, in order of appearance)

The DNA Sequence of PCR Product Denoting Primers and G/T Variants(source Genbank AL391646.12) is as follows:

GAAACTGAGGCTAACAGCTGGGTCTGGCCCATGACTGGTTCAGTTGGTATTTGGTGGACCAAGTTGACCAAGCTCACCCAGCTTCTGAGTGACAGAGTGAATATAAACCCAGCCTGCTCACTCCATTTCCTAGTTTTCTCACCTCTACCAGGGTCTCTGTTGCTCACAAGAGCTCAGGGCTGGGA(T/G)AAGCCTCTGTGTGACAGATGAAGGGGTCCTGCTGCTCTCTAGGGAAGAATCGGTCCCAAATTGCTCAAGGGAGTAAGGTGGTTTCGTTGAGGAGCATCAGCT, identified as SEQ ID No. 9.

When amplicons generated in this way are resolved in an agarose gel, theG-type primers can be shown to have generated a 186-bp product, whereasthe T-type primers give a 132-bp product. The outside primers generate a288-bp product that must be present in every reaction in order toguarantee the reaction has proceeded accurately.

Representative reagents and conditions for the amplification are shownin Table 10.

TABLE 10 PCR conditions for Identification of rs2802292 G-T PolymorphismReagent Final Concentration Vendor AmpliTaq Gold PCR Buffer 1 XPerkin-Elmer dNTPs 200 μM Perkin-Elmer MgCl₂ 1.5 mM Perkin-Elmer“rs2802292_FO” 1.0 μM see above “rs2802292_FI” 1.0 μM see above“rs2802292_RG” 0.5 μM see above “rs2802292_FT” 0.5 μM see above“AmpliTaq Gold” 0.6 U Perkin-Elmer Human DNA 5-10 ng/μL H₂O to volumeThe PCR conditions include 15 minutes at 94° C. followed by 30 cycles of94° C. for 30 seconds, 60° C. for 30 seconds, 72° C. for 30 seconds,then a final incubation at 72° for 7 minutes. The results shown in theexample were performed on an MJ Research model “PTC200” thermocycler.

The amplified fragments can be resolved on a 3% agarose gel as shown inFIG. 1. FIG. 1 gives the results of an ARMS-PCR assay to detect theFOXO3A G/T variants using the primers and conditions described above.Track 1 shows a subject homozygous for the “T” allele (132 bp); tracks 2and 3 show subjects who are homozygous for the “G” allele (186 bp); andtracks 4 and 5 show subjects who are heterozygous for the “T” and “G”alleles (132+186 bp) and M is the 100 bp DNA ladder (Invitrogen,Paisley, United Kingdom).

In summary, FIG. 2 shows a schematic outline of the assay. Primers“rs2802292_FO” and “rs2802292_RO” flank the polymorphic locus rs2802292and should generate a control 288-bp band in all cases. Primers“rs2802292_OF” and “rs2802292_RG” generate a 186-bp G-specific productand primers “rs2802292_FT” and “rs2802292_OR” generate a 132-bpT-specific product.

For use in a kit, oligonucleotides may be any of a variety of naturaland/or synthetic compositions such as synthetic oligonucleotides,restriction fragments, cDNAs, synthetic peptide nucleic acids (PNAs),and the like. The assay kit and method may also employ labeledoligonucleotides to allow ease of identification in the assays. Examplesof labels which may be employed include radio-labels, enzymes,fluorescent compounds, streptavidin, avidin, biotin, magnetic moieties,metal binding moieties, antigen or antibody moieties, and the like.

The kit may, optionally, also include DNA sampling means. DNA samplingmeans are well known to one of skill in the art and can include, but notbe limited to substrates, such as filter papers, the AmpliCard™(University of Sheffield, Sheffield, England S10 2J F; Tarlow, J W, etal., J of Invest. Dermatol. 103:387-389 (1994)) and the like; DNApurification reagents such as Nucleon™ kits, lysis buffers, proteinasesolutions and the like; PCR reagents, such as reaction buffers,thermostable polymerase, dNTPs, and the like; and allele detection meanssuch as the HinfI restriction enzyme, allele specific oligonucleotides,degenerate oligonucleotide primers for nested PCR from dried blood.

C. Pharmacogenomics

Knowledge of the particular alleles associated with a susceptibility todeveloping a particular disease or condition, alone or in conjunctionwith information on other genetic defects contributing to the particulardisease or condition allows a customization of the prevention ortreatment in accordance with the individual's genetic profile, the goalof “pharmacogenomics”. Thus, comparison of an individual's FOXO3Aprofile to the population profile for healthy aging, permits theselection or design of drugs or other therapeutic regimens that areexpected to be safe and efficacious for a particular patient or patientpopulation (i.e., a group of patients having the same geneticalteration).

Knowledge of the particular alleles described in this invention can beused to examine differences in cell behavior in cell cultures and tissuesystems and measure the response of the cells to chemicals or biologicalagents that are added to the cell or tissue culture systems. Differencesin cell behavior and responses can be compared between the genotypes inorder to identify drugs or other pharmacologic agents that may beimplemented in the desire to improve health or extend lifespan or totest new compounds for toxicity or potential effects on genes or geneexpression.

In addition, the ability to target populations expected to show thehighest clinical benefit, based on genetic profile can enable: 1) therepositioning of already marketed drugs; 2) the rescue of drugcandidates whose clinical development has been discontinued as a resultof safety or efficacy limitations, which are patient subgroup-specific;and 3) an accelerated and less costly development for candidatetherapeutics and more optimal drug labeling (e.g. since measuring theeffect of various doses of an agent on the causative mutation is usefulfor optimizing effective dose).

The treatment of an individual with a particular therapeutic agent canbe monitored by measuring the level of expression for a gene associatedwith longevity. The level of expression can be measured by determiningprotein (e.g. FOXO3A), mRNA and/or transcriptional level. Depending onthe level detected, the therapeutic regimen can then be maintained oradjusted (increased or decreased in dose). In a preferred embodiment,the effectiveness of treating a subject with an agent comprises thesteps of: (i) obtaining a pre-administration sample from a subject priorto administration of the agent; (ii) detecting the level or amount of aprotein, mRNA or genomic DNA in the pre-administration sample; (iii)obtaining one or more post-administration samples from the subject afteradministration of the therapeutic agent; (iv) detecting the level ofexpression or activity of the protein, mRNA or genomic DNA in thepost-administration sample; (v) comparing the level of expression oractivity of the protein, mRNA or genomic DNA in the pre-administrationsample with the corresponding protein, mRNA or genomic DNA in thepost-administration sample, respectively; and (vi) altering theadministration of the agent to the subject accordingly.

Cells of a subject may also be obtained before and after administrationof a therapeutic to detect the level of expression of genes other than aFOXO3A gene to verify that the therapeutic does not increase or decreasethe expression of genes which could be deleterious. This can be done,e.g., by using the method of transcriptional profiling. Thus, mRNA fromcells exposed in vivo to a therapeutic and mRNA from the same type ofcells that were not exposed to the therapeutic could be reversetranscribed and hybridized to a chip containing DNA from numerous genes,to thereby compare the expression of genes in cells treated and nottreated with the therapeutic.

The “GCC” haplotype can be used in risk calculators to aid in theprediction of death and age-associated diseases (heart disease, stroke,cancer, COPD or other chronic lung disease, Parkinson disease, anddiabetes and dementia) and future physical function (ability to walk,cognitive function). This information is of interest to the public,physicians, health care companies and insurance companies. Examples ofknown risk calculators include the system and method disclosed in Perls,U.S. Patent Application Publication No. US 2007/0118398 A1, published onMay 24, 2007, which is incorporated by reference herein in its entirety.Risk calculators can be provided in for example, a physician's office,as a handheld or online. An individual, health-care professional,insurance company, health care organization interested in predicting howlong someone will live may enter his/her genotype into a computer andobtains a risk score for aging-related disease, number of healthy yearsof life left, and number of total remaining years of life.

Based on a particular score, a physician or health professional mayadvise the patient on healthy living or risk reduction for the abovediseases and death, particularly for persons with the less protectiveversions of the FOXO3A gene. Some exemplary options include: adviceconcerning food choices (e.g. red wine, soy products, and other foodsthat contain compounds that may affect the activity of the FOXO3A gene)or intensive risk factor modification such as weight loss or increasedphysical activity.

The identification of FOXO3A and in particular the GCC haplotype aspredictors of healthy aging and longevity provides a probable source ofuseful biologics and targets for pharmaceutical screens and testing. Forexample, one may take the gene product or a synthetic version of theprotein or other active compound produced by FOXO3A gene for anticipatedhealth benefits in reduction of age-related diseases. Means of takingthe gene product may include ingestion, injection, transdermaladministration and other methods well known in the pharmaceutical arts.Compounds can be screened to find those that affect the type, activity,or the amount of the gene product produced by FOXO3A, in particular, theGCC haplotype.

The invention includes methods of modulating FOXO3A to prevent or treatage-related diseases. The invention also includes methods for treatingor preventing a disease or condition in which FOXO3A is implicated, e.g.age-related diseases or enhancing longevity in a subject. “Subject,” asused herein, refers to human and non-human animals. The term “non-humananimals” includes all vertebrates, e.g., mammals, such as non-humanprimates (particularly higher primates), farm mammals such as horses,cows, bison, buffalo, goats, pigs and sheep, chicken, ducks and geese,companion animals such as dogs, cats, rabbits, guinea pigs, rodents, andreptiles, and laboratory animals. In a preferred embodiment, the subjectis human. In another embodiment, the subject is an experimental animalor transgenic animal suitable as a disease model. Methods of modulatingand treatment are well known to those skilled in the art as set forth inGeesaman et al., U.S. Patent Application Publication No. US 2007/0105109A1, published on May 10, 2007, which is incorporated by reference hereinin its entirety.

Many other diagnostic and therapeutic uses of the sequences or geneproducts of the allelic variations taught by this invention will beevident to those skilled in the art. Some examples include use in smallmolecule screens, antisense oligonucleotides, double stranded smallinterfering RNAs (siRNAs) will be evident to those skilled in the art.Several approaches to developing diagnostic and therapeutic usesconcerning FOXO activity are described in Goldberg et al., U.S. PatentApplication No. US 2006/0069049 A1, published on Mar. 30, 2006, andconcerning related pathways in Tissenbaum et al., U.S. PatentApplication No. US 2006/0272039, published on Nov. 30, 2006, both ofwhich are incorporated by reference herein in their entirety.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The skilledworker knows, or can identify by using simply routine methods, a largenumber of equivalents of the specific embodiments of the invention.These equivalents are intended to be included in the patent claimsbelow. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description and fall within the scope ofthe appended claims.

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1. A method of diagnosing a predisposition to longevity in a subject,which comprises determining in a tissue sample of said subject whethersaid subject possesses at least one “G” allele of locus/polymorphismrs2802292 at position 109015211 in the FOXO3A gene on chromosome
 6. 2.The method of claim 1, wherein longevity is living at least anadditional 15 years and said predisposition further includes apredisposition to freedom from of at least one chronic diseaseassociated with aging.
 3. The method of claim 2, wherein at least onechronic disease associated with aging is selected from the groupconsisting of diabetes, coronary artery disease and cancer.
 4. A methodof diagnosing a predisposition to longevity in a subject, whichcomprises determining in a tissue sample of said subject whether saidsubject possesses at least one “C” allele of locus/polymorphismrs2764264 at position 109041154 in the FOXO3A gene on chromosome
 6. 5.The method of claim 1, wherein longevity is living at least anadditional 15 years and said predisposition further includes apredisposition to freedom from of at least one chronic diseaseassociated with aging.
 6. The method of claim 2, wherein at least onechronic disease associated with aging is selected from the groupconsisting of diabetes, coronary artery disease and cancer
 7. A methodof diagnosing a predisposition to longevity in a subject, whichcomprises determining in a tissue sample of said subject whether saidsubject possesses at least one “C” allele of locus/polymorphismrs13217795 at position 109080791 in the FOXO3A gene on chromosome
 6. 8.The method of claim 7, wherein longevity is living at least anadditional 15 years and said predisposition further includes apredisposition to freedom from of at least one chronic diseaseassociated with aging.
 9. The method of claim 8 wherein at least onechronic disease associated with aging is selected from the groupconsisting of diabetes, coronary artery disease and cancer
 10. Themethod of claim 1, wherein longevity is living at least an additional 15years and said predisposition further includes a predisposition tofreedom from of at least one chronic disease associated with aging. 11.A method of diagnosing a predisposition to longevity in a subject, whichcomprises determining in a tissue sample of said subject whether saidsubject possesses a “GCC” haplotype, the “GCC” haplotype beingloci/polymorphisms rs2802292, rs2764264, rs13217795, respectively, in aFOXO3A gene.
 12. A method of diagnosing a predisposition to longevity ina non-human subject, which comprises determining in a tissue sample ofsaid subject whether said subject possesses a non-human homologue to a“GCC” haplotype, said “GCC” haplotype being loci/polymorphismsrs2802292, rs2764264, rs13217795, respectively, in a FOXO3A gene.
 13. Akit comprising a first nucleic acid of sufficient length to hybridizesto a first target nucleic acid sequence position selected from the groupconsisting of a “G” allele of locus/polymorphism rs2802292 at position109015211 in the FOXO3A gene on chromosome 6, a “G” allele oflocus/polymorphism rs2802292 at position 109015211 in the FOXO3A gene onchromosome 6 and a “C” allele of locus/polymorphism rs2764264 atposition 109041154 in the FOXO3A gene on chromosome 6, wherein saidfirst nucleic acid hybridizes to said first target nucleic acid sequenceat the position, or the complement of said first target nucleic acidsequence, in one or more containers and instructions for use.
 14. Amethod for testing for the presence of at least one allele of a FOXO3Ahaplotype comprising the steps of: a) collecting a sample of cells froma subject; b) isolating a nucleic acid sample from the cells of thesample; c) contacting the nucleic acid sample with at least one primerwhich specifically hybridizes 5′ and 3′ to at least one allele of aFOXO3A haplotype under conditions such that hybridization andamplification of the allele occurs; and d) detecting the amplificationproduct by allele detection means.
 15. The method according to claim 14,wherein at least one primer is selected from the group consisting of SEQID NOS. 5-8.
 16. A method of monitoring the treatment of a subject witha therapeutic agent by monitoring the level of gene expression of a geneassociated with longevity comprising: a) obtaining a pre-administrationsample from a subject prior to administration of the therapeutic agent;b) detecting the level of gene expression in the pre-administrationsample; c) obtaining one or more post-administration samples from thesubject; d) detecting the level of gene expression in thepost-administration sample; e) comparing the level of gene expression inthe pre- and post-administrative samples to determine the effect of thetherapeutic agent on gene expression; and f) altering the treatment ofthe subject based on the effect of the therapeutic agent on geneexpression.
 17. The method of claim 16, wherein the detecting the levelof gene expression comprises determining at least one of the protein,mRNA or DNA transcription level of the gene associated with longevity.18. The method according to claim 16, wherein the gene associated withlongevity is selected from the group consisting of FOXO3A, LOC100130966,anon-human homologue of FOXO3A, and a non-human homologue ofLOC100130966.
 19. The use of the presence or absence of the “GCC” FOXO3Ahaplotype in a risk calculator to aid in the prediction of death,age-associated diseases or future physical function.